Molecular and genetic studies of antibiotic resistance plasmids in the anaerobic bacterium Bacteroides fragilis were performed in order to analyze genetic exchange mechanisms and the dissemination of antibiotic resistance in this important pathogen. Transposon-like structures encoding clindamycin (Cc) resistance in the three previously described Bacteroides R-plasmids pBF4, pBFTM10, and pBI136 were cloned in E. coli and then characterized by heteroduplex analyses, restriction endonuclease site mapping, and DNA homolgy studies. Results indicated that ll three structures were bounded by a homologous 1.2 kb directly repeated sequence (DRS). The transposon-like elements of pBF4 and pBFTM10 were 5.2 and 5.4 kb in size and shared more than 90% DNA sequence homology. The pBI136 element was 8.4 kb and had diverged significantly; except for the DRS, only 0.85 kb identified as the Cc resistance gene, was homologous to the other two transposon-like elements. This 0.85 kb region was located adjacent to one copy of the DRS. Thus far antibiotic resistance determinants from Bacteroides have not been found to express their phenotype in any other bacterial genus. To facilitate study of antibiotic resistance transfer, a genetic transformation procedure and gene cloning system were developed for B. fragilis. Through the use of these techniques, the pBI136 Cc resistance gene was cloned and expressed in B. fragilis. Analyses of the cloned DNA indicated that the genes does not possess its own promoter sequence but rather it is regulated b y sequences found within the DRS. THese results provide significant insight into the means by which Bacteriodes spp. acquire, regulate, and express new genetic information.